The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice.

Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.     

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.     

The effect of exposure regimen and duration on benzene-induced bone-marrow damage in mice. I. Sex comparison in DBA/2 mice

In the mouse, the concurrent evaluation of micronuclei frequencies in peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) permits an assessment of both recently-induced and chronically-accumulated bone-marrow damage. This assay system was used to evaluate on a weekly basis the effect of exposure duration (1-13 weeks, 6 h per day) and exposure regimen (Regimen 1:5 exposure days per week; Regimen 2:3 exposure days per week) on the ability of 300 ppm benzene to induce genotoxic damage in the bone marrow of male and female DBA/2 mice. In addition, an analysis of the percentage of PCE in peripheral blood was used to evaluate benzene-induced alterations in the rate of erythropoiesis. Exposure to benzene induced a marked increase in the frequency of micronucleated PCE (MN-PCE), an effect which was considerably greater in male mice than in female mice. In both sexes, the induction of MN-PCE was independent of exposure regiment and of exposure duration. Exposure to benzene also resulted in an exposure duration-dependent increase in the frequency of MN-NCE. The frequency of MN-NCE increased more slowly in female than in male mice and, within each sex, more slowly in Regimen 2 animals. Apparent steady-state conditions for MN-NCE frequencies were attained by about the fifth week of exposure in female mice exposed by either regimen and in male mice exposed by Regimen 2. Steady-state conditions for MN-NCE frequencies in male mice exposed to benzene by Regimen 1 did not occur during the duration of the study. An analysis of %PCE data revealed an initial severe depression in the rate of erythropoiesis in both sexes, with the return in the production of PCE to control levels being dependent on both sex and exposure regimen. Suppression of PCE production occurred throughout the course of the study in Regimen 2 males, while the percentage of PCE returned to control levels sporadically after 5 weeks in Regimen 1 males and within 5 weeks in females, regardless of regimen. Thus, while the sex-dependent induction of genotoxic damage by multiple exposures to benzene over a 13-week period was independent of exposure regimen and duration, the induction of cytotoxic damage was both sex- and regimen-dependent. The most severe depression of erythropoiesis occurred in male DBA/2 mice exposed to benzene by the more intermittent regimen (i.e., 3 days/week versus 5 days/week).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of exposure regimen and duration on benzene-induced bone marrow damage in mice. II. Strain comparisons involving B6C3F1, C57B1/6 and DBA/2 male mice

In a companion paper (Luke et al., 1988), the effect of exposure duration and regimen on benzene induced-bone marrow damage was evaluated in male and female DBA/2 mice using the peripheral blood micronucleus assay. To assess the general applicability of the findings obtained for DBA/2 mice to other strains, similar studies were conducted using B6C3F1 and C57B1/6 male mice. An analysis of peripheral blood smears taken weekly from these mice exposed to 300 ppm benzene for 13 weeks (6 h per day) for either 5 days per week (Regimen 1) or for 3 days per week (Regimen 2) revealed: (i) a highly significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE), the magnitude of which was strain specific (DBA/2 greater than C57B1/6 = B6C3F1), but independent of exposure regimen and, except for Regimen 2 B6C3F1 mice, of exposure duration. In male B6C3F1 mice, MN-PCE frequencies increased slightly with increasing exposure duration; (ii) a strain- (C57B1/6 = B6C3F1 greater than DBA/2) and regimen- (Regimen 1 greater than Regimen 2) dependent increase across time in the frequency of micronucleated normochromatic erythrocytes (MN-NCE). Apparent steady-state conditions for MN-NCE frequencies were attained by about 5 weeks of exposure in male mice of all three strains exposed to benzene by Regimen 2. Steady-state conditions for MN-NCE frequencies in male mice exposed to benzene by Regimen 1 did not occur during the duration of the study, with strain-dependent differences in the kinetics of MN-NCE accumulation being present; and (iii) in all 3 strains, an initial severe depression in the rate of erythropoiesis, the return of which to normal levels was both strain- (C57B1/6 = B6C3F1 greater than DBA/2) and regimen- (Regimen 1 greater than Regimen 2) dependent. These data indicate that the induction of genotoxic and cytotoxic damage in the bone marrow of male mice exposed to benzene for 13 weeks can be highly dependent on strain, exposure regimen and exposure duration but that under no circumstance did the level of genotoxic damage induced by benzene decrease under multiple exposure conditions.     

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.     

Hemoglobin adducts in 1,3-butadiene exposed Czech workers: Female–male comparisons

We previously reported results of a molecular epidemiological study of female and male 1,3-butadiene (BD) exposed Czech workers showing that females appeared to absorb or metabolize less BD per unit exposure concentration than did males, based on metabolite concentrations in urine (Chem. Biol. Interact. 166 (2007) 63–77). However, that unexpected observation could not be verified at the time because the only additional BD metabolite measurement attempted was for 1,2,3,4-diepoxybutane (DEB) as reflected in specific N,N[2,3-dihydroxy-1,4-butyl]valine (pyr-Val) hemoglobin adduct concentrations, which were not quantifiable in any subject with the method then employed. Neither somatic gene mutations nor chromosome aberrations were associated with BD exposure levels in that study, consistent with findings in an earlier Czech study of males only. We have since measured production and accumulation of the 1,2-dihydroxy-3,4-epoxybutane (EBD) metabolite as reflected in N-[2,3,4-trihydroxy-butyl]valine (THB-Val) hemoglobin adduct concentrations. The mean THB-Val concentration was significantly higher in exposed males than in control males (922.3 pmol/g and 275.5 pmol/g, respectively), but exposed and control females did not differ significantly (224.5 pmol/g and 181.1 pmol/g, respectively). In both the control and exposed groups mean THB-Val concentrations were significantly higher for males than females. THB-Val concentrations were significantly correlated with mean 8-h TWA exposures for both males and females, but the rate of increase with increasing BD exposure was significantly lower for females. THB-Val concentrations also increased with increasing urine M2 metabolite [isomeric mixture of 1-hydroxy-2-{N-actylcysteinyl}-3-butene and 2-hydroxy-1-{N-acetylcysteinyl}-3-butene] concentrations in both sexes but the rate of increase was also lower in females than in males. There were no significant correlations between THB-Val concentrations and either somatic gene mutations or chromosome aberrations in either males or females. These results using another biomarker to measure a second metabolite of BD support the original conclusion that females absorb or metabolize less BD than males per unit exposure and indicate that the size of the difference increases with exposure. This observation in humans differs from findings in rodents where at prolonged exposures to high BD levels the females form higher amounts of hemoglobin adducts than do males, a difference that disappears at shorter duration lower exposure levels, while female susceptibility to BD induced mutations and tumorgenesis in rodents appears to persist at all BD exposure levels.     

The effects of sublethal concentrations of five organophosphorus insecticides on temperature tolerance, reflexes, and orientation in Gambusia affinis affinis (Pisces: Poeciliidae)

Gambusia affinis were exposed to the insecticides ABATE®, fenthion, chlorpyrifos, methyl parathion, and malathion for 24 h at concentrations of 1, 5, and 10 ppb and for 48 h at 5 ppb. The thermal tolerance was lowered significantly for male and female G. affinis by the toxicants methyl parathion and chlorpyrifos at 5 and 10 ppb with 24 h exposure. Activity was restricted with fish exposed to methyl parathion and chlorpyrifos at concentrations of 10 ppb for 24 h and at 5 ppb for 48 h.     

Exposure assessment of benzene in Thai workers, DNA-repair capacity and influence of genetic polymorphisms

Exposure to benzene can cause DNA damage and the subsequent development of cancer. In this study, study subjects were 31 laboratory workers at a petrochemical factory and 31 gasoline service attendants. Control subjects were 34 workers from a mail sorting service center. Occupational exposures to benzene were assessed using biomarkers of exposure in blood and urine. Induction of DNA-repair capacity was assessed as a biomarker of early effect. The effects of polymorphisms in a metabolizing gene (CYP2E1), in detoxification genes (NQO1 and GSTT1), and in a DNA-repair gene (XRCC1, codon 399) on biomarker levels were evaluated. The mean individual benzene exposure of laboratory workers (24.40+/-5.82 ppb) and that of gasoline service attendants (112.41+/-13.92 ppb) were significantly higher than in controls (1.39+/-0.17 ppb, p<0.001). Blood benzene levels of laboratory workers (169.12+/-30.60 ppt) and gasoline service attendants (483.46+/-59.62 ppt) were significantly higher than those of the controls (43.30+/-4.89 ppt, p<0.001). Trans,trans-muconic acid levels in post-shift urine samples collected from laboratory workers (0.14+/-0.02 mg/g creatinine) and gasoline service attendants (0.20+/-0.02 mg/g creatinine) were significantly higher than in urine samples of controls (0.04+/-0.01 mg/g creatinine, p<0.001). The level of benzene exposure was correlated with blood benzene levels (R2=0.65, p<0.01) and post-shift urinary trans,trans-muconic acid concentrations (R2=0.49, p<0.01). As a biomarker of early effect, DNA-repair capacity was assessed by use of the cytogenetic challenge assay, i.e., chromosomal aberrations in peripheral lymphocytes were assessed after challenging blood cultures with 1 Gy gamma radiation. A significantly lower DNA-repair capacity--determined as dicentrics in laboratory workers (0.17 per metaphase cell) and in gasoline service attendants (0.19 per metaphase cell) compared with controls (0.12 per metaphase cell, p<0.001)--was observed. The frequency of deletions in laboratory workers (0.22 per metaphase cell) and gasoline service attendants (0.39 per metaphase cell) were significantly higher than in control workers (0.16 per metaphase cell, p<0.01 and p<0.001, respectively). An increase in radiation-induced dicentrics and deletions indicate a lower DNA-repair capacity in benzene-exposed workers. The influence of genetic polymorphisms on the biomarkers was assessed. Benzene-exposed workers who carried CYP2E1*1/*5 or *5/*5 genotypes excreted slightly higher levels of trans,trans-muconic acid than workers who carried the CYP2E1*1/*1 genotype. In this study, NQO1 and GSTT1 genotypes did not have any effect on the levels of trans,trans-muconic acid. In the case of XRCC1, laboratory workers with 399Arg/Gln or Gln/Gln had a lower DNA-repair capacity--measured as radiation-induced frequency of dicentrics and deletions--than those with the 399Arg/Arg genotype (p<0.01). Our results show that biomarkers of internal dose and early biological effect in people occupationally exposed to benzene are influenced by genetic polymorphisms in susceptibility genes.     

Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene

Acute myeloid leukemia and chronic lymphocytic leukemia are associated with benzene exposure. In mice, benzene induces chromosomal breaks as a primary mode of genotoxicity in the bone marrow (BM). Benzene-induced DNA lesions can lead to changes in hematopoietic stem cells (HSC) that give rise to leukemic clones. To gain insight into the mechanism of benzene-induced leukemia, we investigated the DNA damage repair and response pathways in total bone marrow and bone marrow fractions enriched for HSC from male 129/SvJ mice exposed to benzene by inhalation. Mice exposed to 100 ppm benzene for 6h per day, 5 days per week for 2 week showed significant hematotoxicity and genotoxicity compared to air-exposed control mice. Benzene exposure did not alter the level of apoptosis in BM or the percentage of HSC in BM. RNA isolated from total BM cells and the enriched HSC fractions from benzene-exposed and air-exposed mice was used for microarray analysis and quantitative real-time RT-PCR. Interestingly, mRNA levels of DNA repair genes representing distinct repair pathways were largely unaffected by benzene exposure, whereas altered mRNA expression of various apoptosis, cell cycle, and growth control genes was observed in samples from benzene-exposed mice. Differences in gene expression profiles were observed between total BM and HSC. Notably, p21 mRNA was highly induced in BM but was not altered in HSC following benzene exposure. The gene expression pattern suggests that HSC isolated immediately following a 2 weeks exposure to 100 ppm benzene were not actively proliferating. Understanding the toxicogenomic profile of the specific target cell population involved in the development of benzene-associated diseases may lead to a better understanding of the mechanism of benzene-induced leukemia and may identify important interindividual and tissue susceptibility factors.     

Does Exposure to Trichloroethene in Low Doses Constitute a Cancer Risk to Humans?

Although chronic exposure to high doses of trichloroethene causes tumors of the lung, liver, and kidney in experimental animals, the epidemiology data in humans exposed to trichloroethene as a whole fail to support a causal association between trichloroethene exposure and cancers of the lungs, liver, or kidneys in humans at environmentally relevant concentrations. Environmentally relevant concentrations of trichloroethene are defined as 50 ppb (50 µg/L) in water or 5 ppb (27 µg/m³) in air. Tumor induction by trichloroethene in rodents exposed to very high doses over their whole lifespan has been observed in the kidney of rats and in the lung and liver of mice. Mechanistic data demonstrate that species-specific processes are involved in the carcinogenicity associated with chronic trichloroethene exposure in rodents. Based on these data and the results of recent well-conducted epidemiology studies, it can be concluded that humans exposed to trichloroethene at environmentally relevant concentrations are not at an increased risk for developing cancer.     

Molecular epidemiological studies in 1,3-butadiene exposed Czech workers: female-male comparisons

Results of a recent molecular epidemiological study of 1,3-butadiene (BD) exposed Czech workers, conducted to compare female to male responses, have confirmed and extended the findings of a previously reported males only study (HEI Research Report 116, 2003). The initial study found that urine concentrations of the metabolites 1,2-dihydroxy-4-(acetyl) butane (M1) and 1-dihydroxy-2-(N-acetylcysteinyl)-3-butene (M2) and blood concentrations of the hemoglobin adducts N-[2-hydroxy-3-butenyl] valine (HB-Val) and N-[2,3,4-trihydroxy-butyl] valine (THB-Val) constitute excellent biomarkers of exposure, both being highly correlated with BD exposure levels, and that GST genotypes modulate at least one metabolic pathway, but that irreversible genotoxic effects such as chromosome aberrations and HPRT gene mutations are neither associated with BD exposure levels nor with worker genotypes (GST [glutathione-S-transferase]-M1, GSTT1, CYP2E1 (5' promoter), CYP2E1 (intron 6), EH [epoxide hydrolase] 113, EH139, ADH [alcohol dehydrogenase]2 and ADH3). The no observed adverse effect level (NOAEL) for chromosome aberrations and HPRT mutations was 1.794 mg/m(3) (0.812 ppm)--the mean exposure level for the highest exposed worker group in this initial study. The second Czech study, reported here, initiated in 2003, included 26 female control workers, 23 female BD exposed workers, 25 male control workers and 30 male BD exposed workers (some repeats from the first study). Multiple external exposure measurements (10 full 8-h shift measures by personal monitoring per worker) over a 4-month period before biological sample collections showed that BD workplace levels were lower than in the first study. Mean 8-h TWA exposure levels were 0.008 mg/m(3) (0.0035 ppm) and 0.397 mg/m(3) (0.180 ppm) for female controls and exposed, respectively, but with individual single 8-h TWA values up to 9.793 mg/m(3) (4.45 ppm) in the exposed group. Mean male 8-h TWA exposure levels were 0.007 mg/m(3) (0.0032 ppm) and 0.808 mg/m(3) (0.370 ppm) for controls and exposed, respectively; however, the individual single 8-h TWA values up to 12.583 mg/m(3) (5.72 ppm) in the exposed group. While the urine metabolite concentrations for both M1 and M2 were elevated in exposed compared to control females, the differences were not significant, possibly due to the relatively low BD exposure levels. For males, with greater BD exposures, the concentrations of both metabolites were significantly elevated in urine from exposed compared to control workers. As in the first study, urine metabolite excretion patterns in both sexes revealed conjugation to be the minor detoxification pathway (yielding the M2 metabolite) but both M1 and M2 concentration values were lower in males in this second study compared to their concentrations in the first, reflecting the lower external exposures of males in this second study compared to the first. Of note, females showed lower concentrations of both M1 and M2 metabolites in the urine per unit of BD exposure than did males while exhibiting the same M1/(M1+M2) ratio, reflecting the same relative utilization of the hydrolytic (producing M1) and the conjugation (producing M2) detoxification pathways as males. Assays for the N,N-(2,3-dihydroxy-1,4-butadyl) valine (pyr-Val) hemoglobin (Hb) adduct, which is specific for the highly genotoxic 1,2,3,4-diepoxybutane (DEB) metabolite of BD, have been conducted on blood samples from all participants in this second Czech study. Any adduct that may have been present was below the limits of quantitation (LOQ) for this assay for all samples, indicating that production of this important BD metabolite in humans is below levels produced in both mice and rats exposed to as little as 1.0 ppm BD by inhalation (J.A. Swenberg, M.G. Bird, R.J. Lewis, Future directions in butadiene risk assessment, Chem. Biol. Int. (2006), this issue). Results of assays for the HB-Val and THB-Val hemoglobin adducts are pending. HPRT mutations, determined by cloning assays, and multiple measures of chromosome level changes (sister-chromatid exchanges [SCE], aberrations determined by conventional methods and FISH) again showed no associations with BD exposures, confirming the findings of the initial study that these irreversible genotoxic changes do not arise in humans occupationally exposed to low levels of BD. Except for lower production of both urine metabolites in females, no female-male differences in response to BD exposures were detected in this study. As in the initial study, there were no significant genotype associations with the irreversible genotoxic endpoints. However, as in the first, differences in the metabolic detoxification of BD as reflected in relative amounts of the M1 and M2 urinary metabolites were associated with genotypes, this time both GST and EH.     

Cytogenetic effects of benzene: dosimetric studies on rats exposed to benzene vapour

Rats were exposed to benzene vapour at nominal concentrations in air of 1, 10, 100 and 1000 ppm acutely for 6 h. Bone marrow cells from each animal were examined for chromosomal abnormalities 24 h after the end of the exposure period. This analysis was carried out on 250 metaphases per animal where possible and showed a significant increase in the percentage of cells with chromosomal abnormalities, excluding gaps, in the groups of animals exposed to 100 and 1000 ppm benzene. In the 10-ppm and 1-ppm exposure groups there were elevated levels of cells with abnormalities which showed evidence of being dose-related, although they were not statistically significant.     

Mutagenicity studies with nitrofurans. I. Mutagenicity of nitrofurylacrylic acid for mammals

Cytogenetic analysis of mouse bone-marrow cells, the dominant lethal test in mice and the cytogenetic analysis of human peripheral lymphocytes in vitro were used to study the mutagenicity of 3-(5-nitro-2-furyl)acrylic acid (5-NFA) for mammals. The bone-marrow cytogenetic analysis was performed in female mice exposed to 5-NFA administered intraperitoneally in single doses of 15--120 mg/kg and in 5 repeated doses of 15 and 30 mg/kg, intragastrically in single doses of 30--240 mg/kg and 5 repeated doses of 30 and 60 mg/kg, and perorally for 12 weeks to 5-NFA concentration of 10, 100 and 1000 mg 5-FNA/1 in drinking water. The bone-marrow analysis was performed in this case after 12 days, 3, 4, 6, 8, 10 and 12 weeks exposure. No increase in chromosome damage attributable to dosing with 5-NFA occurred in any of these experiments. Experiments in which mice were exposed to 5-NFA in drinking water for 12 weeks and then treated with a single i.p. dose of 2 mg of the mutagen TEPA [trix-(1-aziridinyl)phosphine oxide] per kg revealed that, at a concentration of 1000 mg 5-NFA/1, the clastogenic activity of TEPA was reduced to that in untreated animals. The dominant lethal test was performed in male mice exposed to 5-NFA applied intraperitoneally in single doses of 40--120 mg/kg and in 5 repeated doses of 10--30 mg/kg, intragastrically in 5 repeated doses of 20--60 mg/kg, and perorally for 4 weeks in drinking water containing 5-NFA at concentrations of 10, 100, 316 and 1000 mg/l. No significant differences were detected between the exposed and control groups of animals. Experiments in which male mice were exposed to 5-NFA in drinking water and treated after the 4-week exposure to 5-NFA with 1 mg TEPA/kg revealed that a concentration of 1000 mg 5-NFA/1 reduced TEPA-induced dominant lethality to within control values. A reduction in male fertility was observed after the single or repeated 5-NFA doses, but no changes when 5-NFA was applied in drinking water. The cytogenetic analysis of human peripheral lymphocytes exposed in vitro for the last 24 h of culture to concentrations of 1--100 micrograms 5-NFA/Ml did not show any compound-related chromosomal changes. The results of dominant-lethal and bone-marrow cytogenetic studies in mice after consumption of drinking water containing 1000 mg of 5-NFA/1 for 12 weeks and dosed subsequently with TEPA suggests that 5-NFA has some antimutagenic activity. Because none of the studies reported revealed any compound-related genetic activity, the results suggest that 5-NFA is not a chromosome-breaking agent in mammals.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Induction of micronuclei in wild-type and p53(+/-) transgenic mice by inhaled bromodichloromethane

Bromodichloromethane (BDCM) is commonly present in trace amounts in drinking water as a disinfection by-product. BDCM has been shown to be carcinogenic in mice and rats when given by gavage at relatively high doses. Genotoxic activity as well as induced regenerative cell proliferation may contribute to the carcinogenic potential of BDCM. The purpose of the current studies was to evaluate the ability of BDCM to induce micronuclei (MN) in bone marrow and blood of wild-type and p53(+/-) mice on the C57BL/6 and FVB/N genetic backgrounds using the inhalation route of exposure. Toxicity studies were being conducted in this laboratory with inhaled BDCM to select doses for longer-term cancer bioassays using wild-type and p53(+/-) transgenic mice on different genetic backgrounds. Bone marrow samples from these experiments were evaluated for the induction of MN after 1 and 3 weeks of exposure. Accumulation of MN in the peripheral blood was also evaluated at the 13-week time point of a cancer study with the p53(+/-) mice. For the 1-week time point, male C57BL/6 wild-type and p53(+/-) mice and FVB/N wild-type and p53(+/-) mice were exposed daily for 6h per day for 7 consecutive days to atmospheric BDCM concentrations of 0, 1, 10, 30, 100, or 150 ppm. In a second experiment, mice were exposed daily for 6h per day for 3 weeks to atmospheric BDCM concentrations of 0, 0.5, 1, 3, 10, or 30 ppm. Resulting levels of polychromatic erythrocytes (PCE) containing MN were assessed in the bone marrow. For all of the 1- and 3-week exposure groups, the only statistically significant increase in the percentage of bone marrow PCE cells containing MN was in the 1-week 100 ppm BDCM exposure group in the FVB/N wild-type mice (control 0.26% versus exposed 1.16%). C57BL/6 p53(+/-) mice and FVB/N p53(+/-) mice were exposed daily for 6 h per day for 13 weeks to atmospheric BDCM concentrations of 0, 0.5, 3, 10, or 15 ppm. MN were quantified in samples of peripheral blood. Statistically significant increases in the percentage of peripheral blood NCE cells containing MN were seen at the highest BDCM exposure group of 15 ppm in both the C57BL/6 p53(+/-) strain (control 0.36% versus exposed 0.67%) and the FVB/N p53(+/-) strain (control 0.36% versus exposed 0.86%). These data indicate weak induction of MN by BDCM, but only at high atmospheric concentrations relative to normal environmental exposures and with extended periods of exposure. Although comparisons are difficult because responses were negative or marginal, the p53 genotype or the genetic background did not appear to substantially alter susceptibility to the genotoxic effects of BDCM.     

Chromosomic aberrations in female workers exposed to pesticides

The purpose of this work was to determine if the occupational exposure to those pesticides used at banana plantations' packaging plants produces genetic damage to somatic cells of female workers. Chromosomal aberrations were scored in lymphocytes of 20 women, 10 female exposed workers and 10 female controls. Workers were recruited from independent farms from two locations in Costa Rica, during January through June in 1996 and 1997. These females had a minimum of three months of work, had never received chemotherapy or radiotherapy and did some of these labors: sealing, spraying or weighting of bananas. Control unexposed females lived in the same area, were of similar age and neither them nor their husbands/mates had ever worked in pesticide related labors. For each female, 100 mitotic figures were scored. The kind of aberrations detected were acentric fragments, dicentric chromosomes, rings, gaps and breaks. Among workers, 16% of cells (n=1000) had one or more abnormalities, whereas control unexposed females had 6% of cells (n=1000) with comparable anomalies (p < 0.05). In conclusion, the pesticide exposure is a risk factor for chromosome aberrations in female somatic cells.     

Genotoxicity of intermittent co-exposure to benzene and toluene in male CD-1 mice

Benzene is an important industrial chemical. At certain levels, benzene has been found to produce aplastic anemia, pancytopenia, myeloblastic anemia and genotoxic effects in humans. Metabolism by cytochrome P450 monooxygenases and myeloperoxidase to hydroquinone, phenol, and other metabolites contributes to benzene toxicity. Other xenobiotic substrates for cytochrome P450 can alter benzene metabolism. At high concentrations, toluene has been shown to inhibit benzene metabolism and benzene-induced toxicities. The present study investigated the genotoxicity of exposure to benzene and toluene at lower and intermittent co-exposures. Mice were exposed via whole-body inhalation for 6h/day for 8 days (over a 15-day time period) to air, 50 ppm benzene, 100 ppm toluene, 50 ppm benzene and 50 ppm toluene, or 50 ppm benzene and 100 ppm toluene. Mice exposed to 50 ppm benzene exhibited an increased frequency (2.4-fold) of micronucleated polychromatic erythrocytes (PCE) and increased levels of urinary metabolites (t,t-muconic acid, hydroquinone, and s-phenylmercapturic acid) vs. air-exposed controls. Benzene co-exposure with 100 ppm toluene resulted in similar urinary metabolite levels but a 3.7-fold increase in frequency of micronucleated PCE. Benzene co-exposure with 50 ppm toluene resulted in a similar elevation of micronuclei frequency as with 100 ppm toluene which did not differ significantly from 50 ppm benzene exposure alone. Both co-exposures - 50 ppm benzene with 50 or 100 ppm toluene - resulted in significantly elevated CYP2E1 activities that did not occur following benzene or toluene exposure alone. Whole blood glutathione (GSH) levels were similarly decreased following exposure to 50 ppm benzene and/or 100 ppm toluene, while co-exposure to 50 ppm benzene and 100 ppm toluene significantly decreased GSSG levels and increased the GSH/GSSG ratio. The higher frequency of micronucleated PCE following benzene and toluene co-exposure when compared with mice exposed to benzene or toluene alone suggests that, at the doses used in this study, toluene can enhance benzene-induced clastogenic or aneugenic bone marrow injury. These findings exemplify the importance of studying the effects of binary chemical interactions in animals exposed to lower exposure concentrations of benzene and toluene on benzene metabolism and clastogenicity. The relevance of these data on interactions for humans exposed at low benzene concentrations can be best assessed only when the mechanism of interaction is understood at a quantitative level and incorporated within a biologically based modeling framework.     

Acute cytogenetic effect of benzene on rat bone marrow cells in vivo and the effect of inducers or inhibitors of drug-metabolizing enzymes.

Acute cytogenetic effects of benzene in LE rat bone marrow cells in vivo were studied. Chromosome aberrations (CA) induced by benzene consisted mainly of gaps and breaks. Cells with exchanges were rarely observed. The incidence of benzene-induced CA was at its maximum level 12 h after the p.o. or i.p. administration of benzene, dependent on the dose of benzene administered, and higher in male rats than in female rats. However, the sex difference was not observed in the repeated inhalation experiment. Chromosome damage was higher with the p.o. than the i.p. administration. LE rats were more sensitive than Wistar and SD rats to the clastogenic action of benzene. Phenobarbital and Sudan III are well known as inducers of drug-metabolizing enzymes. The peak percentage of benzene-induced CA in the rats pretreated with phenobarbital was observed 6 h after the benzene injection, and it occurred at a higher level than in the rats given only benzene. On the other hand, Sudan III pretreatment suppressed benzene-induced CA at all periods after the benzene injection. SKF-525A (a cytochrome P-450 inhibitor) and cyclohexene oxide (an epoxide hydrase inhibitor) pretreatment also suppressed benzene-induced CA.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

A cytogenetic study of papaya workers exposed to ethylene dibromide

Ethylene dibromide (EDB) has been shown to be carcinogenic in animal studies and mutagenic in vitro. One cytogenetic study of workers exposed to low levels of EDB for short durations was negative. To test whether exposure to low levels of EDB over long periods caused cytogenetic changes, we have assessed the frequencies of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) in the peripheral blood lymphocytes of 60 men occupationally exposed to EDB. These men worked in papaya-packing plants where EDB was used to fumigate the fruit after harvest to kill fruit-fly larvae. 42 other men who worked at a nearby sugar mill served as controls. The average duration of exposure of the papaya workers was 5 years. 82 full shift personal breathing-zone air samples indicated that the papaya workers were exposed to a geometric mean of 88 ppb of EDB, as an 8-h time weighted average (TWA). Peaks up to 262 ppb were measured. The proposed OSHA 8-h TWA for EDB is 100 ppb, while NIOSH recommends 45 ppb. No differences in SCE levels were found between exposed and nonexposed workers. No differences were found in the total CA frequency between exposed and nonexposed workers. SCE levels were significantly increased in men who smoked cigarettes (p = 0.0001) and in men who smoked marijuana (p = 0.01). CA levels showed a significant increasing trend with age (p = 0.03).